1. Field of Use
These teachings relate generally to a system and method for microscopy imaging in general and more particularly to a low numerical aperture (NA) exclusion.
2. Description of Prior Art (Background)
Microscopes have been known for some time in the existing art. Very generally, in upright and inverted light microscopes, focusing of the specimen image is accomplished by way of a corresponding positioning of the specimen relative to the objective, specifically in such a way that a specimen region to be detected is arranged in the focal plane of the objective. This can be achieved on the one hand by the fact that the objective, optionally together with the objective turret receiving the objective, is positioned along the optical axis relative to the specimen. In this case the specimen, for example mounted on a conventional specimen slide, is clamped in a corresponding holder on the microscope stage, this microscope stage then not being moved in the direction of the optical axis of the microscope objective. This type of focusing is usually utilized with inverted light microscopes.
On the other hand, the microscope stage can be arranged movably relative to the microscope stand, and positioned in the direction of the optical axis for focusing. In this case the objective does not perform a motion in the direction of its optical axis relative to the microscope stand. The latter type of focusing is usually utilized with upright light microscopes.
Focusing with the aid of the microscope stage also exists, for purposes of the present invention, when the microscope stage comprises a mechanism with which a specimen slide performs a positioning relative to the objective with the aid of a linear or pivoting motion controlled by a galvanometer, as is the case, for example, with some confocal laser scanning microscopes.
Imaging for scientific research has been evolving since the invention of the microscope. Demand has driven the directions in which these innovations have evolved. In recent years, science has brought the demand for high Z axis resolution to satisfy the need for 3D spatial information in molecular studies. Several technologies have been developed to satisfy this need. Notably, Confocal Microscopy, Two Photon Microscopy, Total Internal Reflection Fluorescence (TIRF) Microscopy, and most recently Stimulated Emission Depletion (STED) Microscopy have come to the forefront. All of these techniques have high resolution imaging in a narrow field of focus.
A narrow field of focus facilitates stacking layers of images called “optical sections” to create high resolution 3D composites of thicker sections. Confocal Microscopy and Two Photon Microscopy are very expensive, but fairly versatile. TIRF Microscopy costs less, has the most discrete Z axis imaging, but is only useful within 200 nanometers of the glass surface thus allowing only one optical section. STED Microscopy is also very expensive, with limited availability.
All of these techniques benefit from the elimination or minimization of the out of focus light, improving the signal to noise ratio. Research has resulted in a demand for mapping trajectories of molecules within cells in 3D. This demand is coupled with the need to image with ever increasing frame rates to provide high resolution 3D position at high temporal resolution. There are several variables which limit the speed of such a system, such as the sensitivity of the detection system (camera, photo-detector, etc.), the speed in which the excitation can scan (Confocal, Two Photon), and the speed in which stage steps in the z axis between scans or images. All of these techniques are effective at excluding unwanted out of focus light. This allows improved imaging in a crowded environment.
Traditional microscopy benefits from a wide depth of field. Depth of field refers to the z axis distance in which an image is in high resolution focus. This is a fundamental property of imaging optics. Cameras have an F-stop which is an adjustable aperture located next to the focusing lens. Some microscope lenses have a similar adjustable aperture. When this aperture is reduced in diameter, less light goes through the lens. More importantly, the light that remains is centered through the middle region on the lens with the exclusion of light near the edges. This condition results in a projected image which is illuminated by light rays which are proportionately more normal in angle to the object and image. Because the angles of these rays are smaller with respect to each other, the focus point is less distinct in the Z axis resulting in a wider range of acceptable focus resolution. An extreme example of this effect is a pinhole camera. In this case the rays are not bent at all by a lens, and thus everything at any distance is in focus. A general observation of this effect would to squint one's eyes in order to read in low light. We benefit in our focus ability from bright light because our pupils are small. A wide aperture passes more light, but has a narrower depth of field. A narrow aperture, passes less light, and produces a wider depth of field. With traditional camera or microscopy configurations when the aperture is open, the depth of field is at its smallest.